Sulfonamides were the first antimicrobial agents effectively used to treat bacterial infections but their use has declined due to the emergence of resistant organisms. Restoration of these approved, well-known drugs could be achieved through inactivation of molecular mechanisms responsible for resistance. We identified a novel mechanism of sulfonamide sensitivity that is caused by a metabolic blockage referred to as the Methyl Folate (MF) trap. Preliminary studies using targeted mutagenesis, genetic and chemical complementation, followed by metabolic analyses, confirmed the MF trap as a novel mechanism of sulfonamide sensitivity. This mechanism is ubiquitously present in mycobacteria and important Gram-negative bacteria such as Pseudomonas aeruginosa, Escherichia coli, and Salmonella typhimurium. Chemical restriction of vitamin B12, required for preventing the MF trap formation, similarly leads to increased sulfonamide susceptibility. The central hypothesis of this application is that the MF trap could be pharmaceutically promoted to render multidrug resistant P. aeruginosa and Enterobacteriaceae to available, clinically approved sulfonamides. The specific aims in the R21 phase are designed to better understand the molecular mechanism by which the MF trap confers sulfonamide sensitivity in bacteria, and whether the MF trap similarly sensitizes drug resistant P. aeruginosa and Enterobacteriaceae to sulfonamides. In Aim 1, genes involved in regulating cellular levels of 5-methyl-tetrahydrolate and homocysteine, the two direct effectors of the MF trap, will be engineered for precise expression control. Cells undergoing titrated gene expression will be simultaneously analyzed for sulfonamide susceptibility and alterations in cellular folate and related metabolites. Furthermore, homocysteinylation of cellular proteins, as a consequence of the MF trap, will be investigated. In Aim 2, P. aeruginosa and S. typhimurium mutants unable to prevent the MF trap will be constructed from multidrug resistant backgrounds, followed by extensive susceptibility tests, both in vitro and during host infections. In addition, we will test the sulfonamide-boosting activiy of a recently developed antivitamin B12 against drug resistant P. aeruginosa and S. typhimurium in macrophages and a larval infection model. We will only proceed with the R33 phase if milestones proposed for the R21 phase are achieved. In Aim 3, series of cobalamin and non-cobalamin anti-B12 molecules will be synthesized to improve efficacy and specificity towards bacterial cells. These compounds will be tested in MF trap-inducing and sulfonamide- boosting assays against both drug susceptible and resistant bacterial strains. Promising compounds will be subjected to in vitro susceptibility testing against a large collection of drug resistant P. aeruginosa and Enterobacteriaceae, as well as to a corneal mouse infection model. In Aim 4, we will assess the effects of these anti-B12 molecules on mammalian B12 and folate metabolism, in order to identify bacterial-specific MF trap inducers that functions as effective SULFA boosters in treating bacterial infections. These proposed studies will set light to a previously unknown mechanism of intrinsic sulfonamide resistance in bacteria. Understanding this mechanism may not only help to improve the clinical use of sulfonamides, but also lead to future development of novel folate antagonistic strategies for drug resistant Gram-negative bacteria.